The present invention relates to a novel DNA coding for D-sorbitol dehydrogenase of a microorganism belonging to acetic acid bacteria including the genus Gluconobacter and the genus Acetobacter, an expression vector containing the said DNA, and recombinant organisms containing the said expression vector. Furthermore, the present invention relates to a process for producing recombinant D-sorbitol dehydrogenase protein and a process for producing L-sorbose by utilizing the said recombinant enzymes or recombinant organisms containing the said expression vector.
L-Sorbose is an important intermediate in the actual industrial process of vitamin C production, which is mainly practiced by Reichstein method (Helvetica Chimica Acta, 17: 311, 1934). In the process, the only microbial conversion is the L-sorbose production from D-sorbitol by Gluconobacter or Acetobacter strains. The conversion is considered to be carried out by NAD/NADP independent D-sorbitol dehydrogenase (SLDH). L-Sorbose is also a well known substrate in the art for microbiologically producing 2-keto-L-gulonic acid, which is a useful intermediate in the production of vitamin C.
It is known that there are NAD/NADP-independent D-sorbitol dehydrogenases which catalyze the oxidation of D-sorbitol to L-sorbose. One such D-sorbitol dehydrogenase was isolated and characterized from Gluconobacter suboxydans var .alpha. IFO 3254 (E. Shinagawa et al., Agric Biol. Chem., 46: 135-141, 1982), and found to consist of three subunits with the molecular weight of 63 kDa, 51 kDa, and 17 kDa; the largest subunit is dehydrogenase containing FAD as a cofactor, the second one is cytochrome c and the smallest one is a protein with unknown function; and shows its optimal pH at 4.5. Such SLDH was also purified and characterized from G. suboxydans ATCC 621 (KCTC 2111) (E-S Choi et al., FEMS Microbiol. Lett. 125:45-50, 1995) and found to consist of three subunits with the molecular weight of 75 kDa, 50 kDa, and 14 kDa; the large subunit is dehydrogenase containing pyrroloquinoline quinone (PQQ) as a cofactor, the second one is cytochrome c and the small one is a protein with unknown function. The inventors also purified and characterized the NAD/NADP-independent SLDH from G. suboxydans IFO 3255 (T. Hoshino et al., EP 728840); the SLDH consists of one kind of subunit with the molecular weight of 79.0 +/-0.5 kDa and shows its optimal pH at 6 to 7 and shows dehydrogenase activity on mannitol and glycerol as well as on D-sorbitol.
Although several SLDHs have been purified, their genes have not been cloned yet. It is useful to clone the SLDH gene for efficient production of the SLDH enzyme and for constructing recombinant organism having enhanced SLDH activity to improve the production yield of L-sorbose. It is also useful to introduce the said SLDH gene into desired organisms, for example, Gluconobacter converting L-sorbose to 2-keto-L-gulonic acid for constructing recombinant microorganisms which directly produce 2-keto-L-gulonic acid from D-sorbitol.